The rise in antibiotic-resistant Mycobacterium tuberculosis and the lack of drugs capable of efficiently eradicating persistent bacilli responsible for life-long infections in humans emphasize the need for novel anti- TB agents with mechanisms of action different from those of existing drugs. The search for alternative drug therapies has led to our interest in class II fructose-1, 6-bisphosphate aldolase (FBA), a key enzyme of glycolysis/gluconeogenesis that was shown to be induced in M. tuberculosis grown under oxygen-limiting conditions thought to mimic the physical microenvironment encountered by persistent bacilli in pulmonary lesions. Selective inhibition of FBA is expected to prevent M. tuberculosis from growing on host-derived fatty acids during persistent infection. Although ubiquitous in living organisms, FBAs can be divided into two classes which differ in their structure and reaction mechanism. While class I FBAs are the only type found in mammals, prokaryotes produce class II FBAs. The absence of class II FBAs from mammalian cells and the specificity of their structure and catalytic mechanism should make it possible to design specific inhibitors of class II enzymes that target pathogenic bacteria without affecting the host's gluconeogenetic and glycolytic pathways. The broad goal of this research project is thus the development of a high-throughput assay for FBA of M. tuberculosis (FBA-tb) and its use to identify inhibitors of this enzyme. Such inhibitors will not only provide bases for the rational design of drugs targeting persistent M. tuberculosis but also provide us with tools which we will use to establish the physiological function of FBA-tb in actively-replicating and non-replicating bacilli in vivo and in vitro models of TB persistence. PUBLIC HEALTH RELEVANCE: Novel drug targets whose inhibition would result in the killing of multi-drug-resistant as well as persistent Mycobacterium tuberculosis are urgently needed. We propose to develop of a high-throughput assay for the class II fructose 1, 6 bisphosphate aldolase of Mycobacterium tuberculosis and to use this assay to identify inhibitors effective against persistent bacilli.